Human Genome Project Gene Therapy

1. Human Genome ProjectGene Therapy Chapter 10 BIT 120

2. Human Genome Project • Begun in 1990, the U.S. Human Genome Project is a 13-year effort coordinated by the Department of Energy and the National Institutes of Health. • Human Genome Project video

3. Goals of the Project • identify all the approximately 30,000 genes in human DNA, • determine the sequences of the 3 billion chemical base pairs that make up human DNA, • store this information in databases, • improve tools for data analysis, • transfer related technologies to the private sector, and • address the ethical, legal, and social issues (ELSI) that may arise from the project.

4. Genetic Disease and Gene Therapy • 5 to 10% of children inherit an identifiable genetic defect • can alleviate symptoms, but cure the disease? • many diseases all about genetics- hemophilia

5. Genetic Disease • others a combination- FH- familial hypercholesterolemia- defect in LDL receptor gene- does not take up LDL and process it, so large amounts found in blood. • rigorous diet and exercise can improve the lipid profile of someone with FH • look at inheritance pattern and pedigree analysis

6. Finding a disease Gene • Finding and cloning a disease causing gene (in the absence of all information- no probe from an animal sequence, etc.) • How does one go about it?

7. Cystic Fibrosis • CF –cystic fibrosis- most common lethal autosomal disease among Caucasians • Abnormal glandular secretions that cause pulmonary, digestive, pancreatic and dysfunction • Respiratory track infections and malnutrition (inability to digest food) • One in every 1800 Caucasian children • Rarely live past age 40- 50% die before age 21

8. Some techniques used • Southern Blot- DNA on gel with a DNA probe – named after discoverer (E.M. Southern) • Northern Blot –RNA on gel with a DNA probe • Western- proteins with antibodies

9. CF and Genetic Linkage • Closer two genes are to each other on the chromosome, the more likely they are to remain together during meiosis (genetic linkage) • So, first phase was to establish a linkage between CF and some genetic marker • Figures 10.2 and 10.3- pages 210-211 • Also go over Roche Genetics exercise

10. CF discovery (cont’d) • How do you know you have reached CF gene in chromosomal walk? • You look for ORF within each newly cloned sequence. Any ORF they found could be the CF gene. • look at mRNAs of patients with CF; chose sweat glands, known to be affected in CF patients. – isolated sample from sweat glands and probed mRNA with fragment of DNA – looked for match

11. Proof they had CF gene • one gene identified that is expressed differently in CF patients than in normal patients. • Mutation found in every CF gene patients studied- not found in normal patients (looked at many patients) • Chloride transport – deficient in secretory cells from CF patients. Cultured their cells in lab, put a normal copy of CF gene in, restored chloride ion transport.- should really be CF gene. • Technique also called positional cloning

12. Problems with technique • More than one mutation can cause a defect • Conditions need to be right to detect a single base pair change • Salt and temperature conditions for the hybridization • Develop a specific probe – like one in 70% CF patients • Over 100 alleles of CF – can miss some

13. Genetic Diagnosis • Determining whether or not a particular gene defect is present in an individual. • Genetic diagnosis – adults – e.g. Huntington’s disease • Embryonic diagnosis –in vitro fertilization; implant disease free embryos • Prenatal Diagnosis – done before baby is born • Amniocentesis • CVS (chorionic villus sampling)

14. Treatment (Gene Therapy) • Gene Therapy to take place if (p.225) • 1. Gene to be transferred is available • 2. Effective method of introducing the gene into cells is necessary • 3. Target tissue or cells accessible to gene therapy • 4. Procedure does no harm to patient • 5. Treatment significantly improves health of patient

15. Gene Therapy • Transgenic humans • Use retroviruses as vectors • Things needed for procedure to work • Small inserts – need to accept non viral gene • Need to inactivate virus genes – no new viruses to be produced • Cells must be dividing – to incorporate DNA

16. Gene Therapy • Figure 10.7, p. 227 – replicative cycle of a retrovirus • Ex-vivo gene therapy – Figure 10.8, p.228 • Conditions needed: • Take bone marrow cells out of the body • Insert retrovirus with gene of interest • Select for ones with gene • Put back into marrow or blood (diseases that affect the blood good targets for ex vivo gene therapy)

17. Problems with Gene Therapy • Problems with ex-vivo method • Not enough cells get desired gene to correct problem • Modified cells don’t last long; need repeat treatments

18. Risks • Other risks with gene therapy: - Cells injected may cause an immune response • Random insertion of retrovirus into host chromosome- may be likely in non-coding DNA, but what if it interrupts the coding DNA? • Too much of a good thing- too much clotting factor causing unwanted clots

19. Gene Therapy • Table 10.1 – diseases may benefit from gene therapy

20. Germ line gene therapy • can’t deliver to a specific location in the chromosome- what would random insertion cause?? – been OK so far with transgenic animals – lots of noncoding DNA to accept gene • few would survive genetic manipulation, fertilization, selection and implantation